The focus of this work has been to understand the molecular details that control the interaction of MHC molecules with NK receptors, T cell receptors, and with coreceptors such as CD8. These studies are dependent upon both functional and biophysical analysis of the interaction of the molecules in question, and attempts are made to correlate binding properties with function and structure. Our most exciting progress in the past year has been the development of a general model system for both the engineering of NK receptors of the C- type lectin family, and the use of these in biophysical binding studies. These experiments are of significant interest and importance to a basic understanding of immune recognition because they allow the characterization of the molecular details of a family of cell-cell interactions that reflect the innate rather than the adaptive immune system. We have engineered and isolated NK receptors, Ly49A, Ly49C, and Ly49G2, representatives of the inhibitory receptors of the C-type lectin family. To date, most of our experience is with Ly49A, which we have shown is produced as a non-covalent dimer, that binds H-2Dd in the absence of Ca++ (indicating that this is not a bona fide C-type lectin), that binds bacterial H-2Dd (which lacks carbohydrate) indicating that carbohydrate is not required for the interaction. We have characterized the lack of peptide specificity of the interaction, and using both analytical ultracentrifugation and surface plasmon resonance we have been able to measure the affinity of interaction of Ly49A with H-2Dd. The ability to bind H-2Dd resides in a fragment of the Ly49A molecule that extends from amino acid 127 to the terminus at 262, the fragment that we have crystalized. The affinity of the Ly49A for H-2Dd as determined at 25?C by surface plasmon resonance in many determinations ranges from 6.1 to 26 micromolar, and is characterized by association rate constant ka of 1300 to 5100 M-1s-1 and dissociation rate constant kd of 0.024 to 0.031 s-1. These parameters are remarkably similar to those that we have observed in the binding of H-2Dd to a cognate T cell receptor. The most interesting observation of our binding studies is that in multiple component binding assays we demonstrate the ability of H-2Dd to simultaneously interact with both the cognate T cell receptor and the Ly49A NK receptor. This has important biological implications in that a number of bona fide T cells have been identified as expressing both T cell receptors as well as NK receptors. If a T cell bearing an inhibitory or activating NK receptor can bind the same MHC molecule simultaneously, there is the opportunity for complex regulatory signaling based on these interactions. Additional experiments in this project have included collaborations to exploit other biophysical methods to examine the interaction of peptides with MHC molecules. To this end, we have collaborated with the laboratory of Dr. I. Pecht in the analysis by fluorescence spectroscopy of the interaction of H-2Kb-restricted peptides. Analysis of these data lead to the consistent conclusion that the MHC molecule changes its shape on binding peptides, and that this follows a complex kinetic pathway. - MHC, NK receptors, T cell receptors, peptide, molecular interactions.